Biomarkers, such as hormones, vitamins, and/or metabolites, can be used for the clinical diagnosis of multiple disorders and as endogenous biomarkers in endocrinology. Steroid hormones, such as delta 5 steroids, are an important class of hormones.
Requirements for the clinical diagnostic testing of endogenous biomarkers in endocrinology may include highly sensitive and specific assays, the ability to analyze small sample volumes (e.g., pediatric sample volumes can be limited to less than about 200 μL), and the ability to screen for multiple analytes to accurately diagnose a disease state, e.g., an endocrine disorder. Historically, radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods have been used in such clinical diagnostic testing. Immunoassay methods (IA), such as RIA and EIA, however, may suffer from low throughput, antibody cross-reactivity, which can require extra preparation for specificity, and poor scalability. Also, the analysis of endogenous biomarkers by RIA may require multiple serial dilutions for the analysis of each individual marker, which can lead to the need to make multiple adjustments to normalize sample volumes and/or the need for multiple separate tests. Also, immunoassay testing is not particularly conducive to the analysis of multiple biomarkers in each sample. The analysis for multiple analytes in a single assay can allow for using samples of reduced size which results in assays of increased sensitivity and efficiency per sample.
Thus, there is a need to develop analytical techniques that can be used for the measurement of endogenous biomarkers, and for methods that provide more sensitivity and higher throughput than RIA. Until recently, however, only GC-MS or LC-MS/MS with derivatization has been successful for small sample volumes. Thus, there is a need in the art for LC-MS/MS techniques for the analysis of endogenous biomarkers for clinical diagnosis in endocrinology capable of providing detection limits at acceptable levels, without the need for the cumbersome derivatization processes.